RESUMEN
Recent studies have shown that aldosterone may play a critical role in the transition to heart failure and that heart is a direct target of the action of aldosterone, which can provoke hypertrophy and apoptosis of isolated cardiomyocytes and also increase the expression of genes that favor tissue fibrosis. Early work from this and other laboratories has established a link between the aliphatic polyamines and cardiac hypertrophy, while more recently an involvement of polyamines even in cell death and survival has emerged. In the present study we have treated cardiac cells, i.e. rat H9c2 cardiomyoblasts and neonatal cardiomyocytes, with (D, L)-2-(difluoromethyl)ornithine, a specific inhibitor of polyamine biosynthesis, to investigate the effects of polyamines in relation to the hypertrophic, pro-fibrotic and pro-apoptotic actions of aldosterone. The results indicate that inhibition of polyamine biosynthesis may prevent or attenuate the adverse actions of aldosterone, by modulating the expression of genes related to cardiac hypertrophy and fibrosis, as well as the levels of proteins and the activities of enzymes that control apoptosis.
Asunto(s)
Aldosterona/farmacología , Eflornitina/farmacología , Cardiopatías/patología , Miocitos Cardíacos/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Poliaminas Biogénicas/biosíntesis , Células Cultivadas , Eflornitina/química , Fibrosis/metabolismo , Expresión Génica/efectos de los fármacos , Cardiopatías/tratamiento farmacológico , Cardiopatías/metabolismo , Cardiopatías/fisiopatología , Hipertrofia/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Ratas , Ratas WistarRESUMEN
Growing evidence suggests a role for polyamines in apoptosis, although the relationship appears to be complex. alpha-Difluoromethylornithine (DFMO), a largely used ornithine decarboxylase inhibitor, is cytostatic, hardly cytotoxic and may even increase the resistance of tumour cells to some apoptotic stimuli. This may represent a problem in cancer therapy, where the killing of tumoral cells would be a desired effect, but could be an advantage in other pathological contexts related to an excess of apoptosis, such as cardiovascular diseases, stem cell transplantation, arthritis and infections. In different cellular models, polyamine depletion following treatment with polyamine biosynthesis inhibitors appears to inhibit mitochondrial and death receptor pathways of apoptosis by affecting key proteins. These studies indicate that inhibition of polyamine biosynthesis may prevent or reduce the apoptotic response triggered by a variety of stimuli in non-tumoral cells, such as cardiac cells, stem cells, chondrocytes, macrophages and intestinal epithelial cells.
Asunto(s)
Apoptosis/efectos de los fármacos , Poliaminas Biogénicas/biosíntesis , Amidinas/farmacología , Animales , Caspasas/efectos de los fármacos , Caspasas/metabolismo , Supervivencia Celular/efectos de los fármacos , Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , Eflornitina/farmacología , Humanos , Indanos/farmacología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Inhibidores de la Ornitina Descarboxilasa , Péptido Hidrolasas/metabolismo , Putrescina/metabolismo , Transducción de Señal/efectos de los fármacos , Espermidina/metabolismoRESUMEN
Intracellular signaling pathways involved in the survival of proliferating L1210 leukemia cells were investigated by using specific modulators. Among the various inhibitors tested, only 1H-[1,2,4]oxadiazole [4,3-a]quinoxalin-1-one (ODQ), a soluble guanylate cyclase (sGC) inhibitor, was found to induce a marked increase in caspase activity, which was associated with a loss of cell viability and a reduction in cGMP content. ODQ also provoked the processing of caspases-3 and -9, release of cytochrome c and, as early events, reduction of Bcl-2 content and dephosphorylation of Bad at Ser 112. Furthermore, YC-1, an sGC activator, and 8-Br-cGMP, a cell-permeant analogue of cGMP, exerted some protection against various apoptotic stimuli, such as serum deprivation or spermine accumulation. Although PD98059 (2'-amino-3'-methoxyflavone), an inhibitor of the p44/42 mitogen-activated protein kinase (MAPK) pathway, did not increase basal caspase activity, and ODQ did not affect p44/42 MAPK phosphorylation significantly, phorbol myristate acetate stimulated p44/42 MAPK and reduced caspase activation induced by ODQ, serum deprivation, and spermine in a p44/42-dependent manner. SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)1H-imidazole), a p38 MAPK inhibitor, also partially protected against ODQ-induced apoptosis by increasing p44/42 MAPK phosphorylation. In conclusion, these results suggest that sGC may be relevant both for survival of L1210 cells under basal growing conditions and for protection against various apoptotic stimuli. p44/42 MAPK activation may also confer some protection from apoptosis, but apparently through a pathway largely independent of cGMP.
Asunto(s)
Guanilato Ciclasa/fisiología , Leucemia L1210/patología , Proteína Quinasa 1 Activada por Mitógenos/fisiología , Proteínas Quinasas Activadas por Mitógenos/fisiología , Animales , Apoptosis , Caspasas/metabolismo , División Celular/efectos de los fármacos , División Celular/fisiología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , GMP Cíclico/metabolismo , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Guanilato Ciclasa/antagonistas & inhibidores , Imidazoles/farmacología , Ratones , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Oxadiazoles/farmacología , Oxidación-Reducción , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Piridinas/farmacología , Quinoxalinas/farmacología , Transducción de Señal , Células Tumorales CultivadasRESUMEN
Activation of the caspase proteases represents a central point in apoptosis. The requirement for spermine for the processes leading to caspase activation has been studied in transformed embryonic fibroblasts obtained from gyro (Gy) mutant male mice. These cells lack spermine synthase activity and thus provide a valuable model to study the role of spermine in cell processes. Gy fibroblasts do not contain spermine and have a higher spermidine content. However, when compared with fibroblasts obtained from normal male littermates (N cells), Gy fibroblasts were observed to grow normally. The lack of spermine did not affect the expression of Bcl-2, and caspases 3 and 9 were activated by etoposide in both N and Gy cells, indicating that spermine is dispensable for caspase activation. Spermine deficiency did not significantly influence caspase activity in cells treated with etoposide, cycloheximide or staurosporine, but sensitized the cells to UV irradiation, which triggered significantly higher caspase activity in Gy cells compared with N cells. alpha-Difluoromethylornithine (DFMO), an inhibitor of polyamine synthesis that is able to deplete cells of putrescine and spermidine, but usually does not influence spermine content, was able to produce a more complete polyamine depletion in Gy cells. This depletion, which included spermine deficiency, dramatically increased caspase activation and cell death in Gy fibroblasts exposed to UV irradiation. On the other hand, in either N or Gy cells, DFMO treatment did not influence caspase activity triggered by staurosporine, but inhibited it when the inducers were cycloheximide or etoposide. In Gy cells depleted of polyamines by DFMO, polyamine replenishment with either spermidine or spermine was sufficient to restore caspase activity induced by etoposide, indicating that, in this model, polyamines have an interchangeable role in supporting caspase activation. Therefore, spermine is not required for such activation, and the effect and specificity of polyamine depletion on caspase activity may be very different, depending on the role of polyamines in the specific death pathways engaged by different stimuli. Some inducers of apoptosis, for example etoposide, absolutely require polyamines for caspase activation, yet the lack of polyamines, particularly spermine, strongly increases caspase activation when induced by UV irradiation.
Asunto(s)
Caspasas/metabolismo , Poliaminas/metabolismo , Espermina Sintasa/metabolismo , Animales , Western Blotting , Células Cultivadas , Cicloheximida/farmacología , Eflornitina/farmacología , Activación Enzimática , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Fibroblastos/metabolismo , Masculino , Ratones , Ratones Mutantes , Inhibidores de la Síntesis de la Proteína/farmacología , Espermina Sintasa/genéticaRESUMEN
Cytochrome c release from mitochondria to the cytosol represents a critical step in apoptosis, correlated to the activation of the caspase cascade. In this report, we show that addition of micromolar concentrations of polyamines to isolated rat heart mitochondria induces the release of cytochrome c. Spermine, which is effective at concentrations of 10-100 microM, is more potent than spermidine, whereas putrescine has no effect up to 1 mM. The release of cytochrome c caused by spermine is a rapid, saturable and selective process that is independent of mitochondria damage. Spermine, unlike polylysine, is able to release a discrete amount of cytochrome c from intact, functional mitochondria. The cytochrome c-releasing power of spermine is not affected by cyclosporin A, differently from the effect of permeability transition inducers. In a cardiac cell-free model of apoptosis, the latent caspase activity of cytosolic extracts from cardiomyocytes could be activated by cytochrome c released from spermine-treated heart mitochondria. These data indicate a novel mechanism of cytochrome c release from the mitochondrion, and suggest that prolonged and sustained elevation of polyamines, characteristic of some pathologies such as heart hypertrophy, could be involved in the development of apoptosis.
Asunto(s)
Grupo Citocromo c/metabolismo , Mitocondrias Cardíacas/efectos de los fármacos , Mitocondrias Cardíacas/metabolismo , Poliaminas/farmacología , Animales , Apoptosis , Caspasas/metabolismo , Extractos Celulares , Embrión de Pollo , Ciclosporina/farmacología , Citosol/efectos de los fármacos , Citosol/enzimología , Citosol/metabolismo , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Femenino , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/metabolismo , Cinética , Miocardio/citología , Permeabilidad/efectos de los fármacos , Polilisina/farmacología , Putrescina/farmacología , Ratas , Ratas Wistar , Espermidina/farmacología , Espermina/farmacologíaRESUMEN
Caspase enzymes are a family of cysteine proteases that play a central role in apoptosis. Recently, it has been demonstrated that caspases can be S-nitrosylated and inhibited by nitric oxide (NO). The present report shows that in chick embryo heart cells (CEHC), NO donor molecules such as S-nitroso-N-acetylpenicillamine (SNAP), S-nitrosoglutathione, spermine-NO or sodium nitroprusside inhibit caspase activity in both basal and staurosporine-treated cells. However, the inhibitory effect of NO donors on caspase activity is accompanied by a parallel cytotoxic effect, that precludes NO to exert its antiapoptotic capability. N-Acetylcysteine (NAC) at a concentration of 10 mM blocks depletion of cellular glutathione and cell death in SNAP-treated CEHC, but it poorly affects the ability of SNAP to inhibit caspase activity. Consequently, in the presence of NAC, SNAP attenuates not only caspase activity but also cell death of staurosporine-treated CEHC. These data show that changes in the redox environment may inhibit NO-mediated toxicity, without affecting the antiapoptotic capability of NO, mediated by inhibition of caspase enzymes. NO may thus be transformed from a killer molecule into an antiapoptotic agent.
Asunto(s)
Apoptosis/fisiología , Inhibidores de Caspasas , Corazón/fisiología , Óxido Nítrico/fisiología , Acetilcisteína/farmacología , Animales , Apoptosis/efectos de los fármacos , Células Cultivadas , Embrión de Pollo , Glutatión/análogos & derivados , Glutatión/farmacología , Corazón/embriología , Óxido Nítrico/antagonistas & inhibidores , Nitrocompuestos/farmacología , Penicilamina/análogos & derivados , Penicilamina/farmacología , Estaurosporina/farmacologíaRESUMEN
The purpose of the study is to present the percentile distribution of body mass index in Zagreb school children and to assess whether it differs from those in other countries; in addition, to assess whether the values of mean BMI in Zagreb school children differ markedly from those in other regions in Croatia i.e. in Medimurje and Osijek. Data on height and weight have been derived from growth surveys organized or performed by the Andrija Stampar School of Public Health over the last decade. The results have shown the skewed distribution of data i.e. the agglomeration of high values of BMI in upper percentile positions. Percentile values of BMI for Zagreb school children were higher than the reference data for American white children and adolescents except at the upper percentile positions (85 and 95) for the older age groups. Mean values of BMI of Zagreb school boys and girls were in general higher than in their peers in other European countries. However, the means of BMI for two other groups of Croatian children--in Osijek and Medimurje--were lower. Regarding the association of overweight with risk factors for cardiovascular diseases, the results have pointed out a great importance of the respective health education programme for school children and adolescents. The presented results may also serve as a basis for a study of secular changes in variations of body mass index in the adolescent period.
Asunto(s)
Índice de Masa Corporal , Adolescente , Antropometría , Niño , Fenómenos Fisiológicos Nutricionales Infantiles , Croacia , Femenino , Humanos , MasculinoRESUMEN
Polyamines are ubiquitous organic cations required for cell proliferation. However, some evidence suggested that their excessive accumulation can induce apoptosis. We show here that, in a post-nuclear extract from U937 cells, the addition of spermine triggers the death program, represented by cytochrome c exit from mitochondria, the dATP-dependent processing of pro-caspase-3 and the onset of caspase activity. Spermine is more effective than spermidine, whereas putrescine has no effect. Polyamine acetylation abolishes their pro-apoptotic power. These data demonstrate a direct mechanism responsible for polyamine toxicity and also suggest that an excessive elevation of free polyamines could be involved in the transduction of a death signal.
Asunto(s)
Apoptosis , Caspasas/fisiología , Espermina/farmacología , Espermina/fisiología , Animales , Caspasa 3 , Sistema Libre de Células , Relación Dosis-Respuesta a Droga , Humanos , Mitocondrias/enzimología , Miocardio/enzimología , Poliaminas/metabolismo , Ratas , Factores de Tiempo , Células U937RESUMEN
Exposure of several leukaemia cell types to the polyamine spermine triggered caspase activation. In HL60 cells, the onset of caspase activity correlated with the accumulation of spermine, and was accompanied by the processing of the caspase-3 precursor and the digestion of the substrate proteins PARP and gelsolin. Spermine also induced the accumulation of cytochrome c in the cytosol. Caspase activation triggered by spermine was not blocked by antioxidants or inhibition of polyamine oxidase. The deregulation of polyamine uptake strongly sensitised the cells to spermine-induced caspase activation. These data show that an excessive intracellular level of spermine triggers caspase activation that is not mediated by oxidative mechanisms, and suggest a model where elevated free cytosolic polyamines may act as transducers of a death message.
Asunto(s)
Caspasas/metabolismo , Leucemia/enzimología , Espermina/farmacología , Animales , Caspasa 3 , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Activación Enzimática/efectos de los fármacos , Células HL-60 , Humanos , Células Jurkat , Leucemia L1210 , Poliaminas/metabolismo , Células U937RESUMEN
The ability of natural and synthetic opioids to modulate the induction of ornithine decarboxylase (ODC) was investigated in immune cells and cardiomyocytes in culture. In particular, Leu-enkephalin, which shows preference for delta-receptors, enhanced ODC activity in both thymocytes and cardiomyocytes, whereas the effect of U-50488H, a synthetic kappa-selective agonist, was cell-specific. In thymocytes, U-50488H markedly inhibited the induction of the enzyme elicited by the mitogen concanavalin A (Con A) or by a combined treatment with PMA and A23187, and also reduced basal ODC activity. However the drug did not affect ODC induced by other stimuli. The inhibition of the induction of ODC activity was accompanied by a reduction of ODC mRNA level and an acceleration of ODC turnover. The action of U-50488H in thymocytes does not appear to be mediated by kappa or other classical opioid receptors lacking both stereospecificity and antagonist sensitivity, but may involve a pertussis toxin-sensitive G protein. Splenocytes also showed the ODC inhibiting effect of U-50488H, although they were less sensitive compared to thymocytes. In contrast, U-50488H enhanced ODC activity in cardiomyocytes and this effect was blocked by a specific kappa-antagonist. In conclusion, these results indicate that some opioid agonists can modulate ODC expression in non neural cells. In particular, kappa-opioid receptors may be involved in the U-50488H action in cardiomyocytes, and a distinct site, linked to inhibition of cell proliferation, may operate in immune cells.
Asunto(s)
Ornitina Descarboxilasa/biosíntesis , Receptores Opioides/agonistas , 3,4-Dicloro-N-metil-N-(2-(1-pirrolidinil)-ciclohexil)-bencenacetamida, (trans)-Isómero/farmacología , Analgésicos no Narcóticos/farmacología , Animales , Linfocitos B/citología , Linfocitos B/efectos de los fármacos , Células Cultivadas , Encefalina Leucina/farmacología , Inducción Enzimática/efectos de los fármacos , Sistema Inmunológico/citología , Sistema Inmunológico/efectos de los fármacos , Ligandos , Masculino , Miocardio/citología , Miocardio/metabolismo , Especificidad de Órganos , Ornitina Descarboxilasa/efectos de los fármacos , ARN Mensajero/biosíntesis , ARN Mensajero/efectos de los fármacos , Ratas , Ratas Wistar , Linfocitos T/citología , Linfocitos T/efectos de los fármacosRESUMEN
Recent investigations have indicated the involvement of proteasome in programmed cell death. The present studies show that although peptide aldehyde inhibitors of proteasome are by themselves weak inducers of apoptosis, they inhibit the apoptotic effect of the anticancer drug etoposide in rat thymocytes. Acetyl-Leu-Leu-norvalinal (LLnV-al) and other related peptide aldehydes inhibited the increase in caspase activity and DNA fragmentation that followed treatment with etoposide and their effect was related to their potency as proteasome inhibitors. To inhibit etoposide-induced apoptosis, LLnV-al must be present within 3 h of treatment with etoposide, in the same way as the inhibitor of protein synthesis cycloheximide must be. Etoposide caused a rapid accumulation of p53 protein that was not inhibited by LLnV-al, which was also a strong inducer of p53. Peptide aldehydes were also weak activators of caspase activity, suggesting that the same mechanism, i.e. the blocking of proteasome function, both triggers apoptosis and inhibits the effect of etoposide. These results are consistent with a model in which proteasome is selectively involved in the pathway used by etoposide to induce cell suicide.
Asunto(s)
Aldehídos/farmacología , Antineoplásicos Fitogénicos/toxicidad , Apoptosis/efectos de los fármacos , Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Etopósido/toxicidad , Complejos Multienzimáticos/metabolismo , Oligopéptidos/farmacología , Animales , Apoptosis/fisiología , Fragmentación del ADN/efectos de los fármacos , Técnicas In Vitro , Linfocitos/efectos de los fármacos , Linfocitos/fisiología , Masculino , Complejo de la Endopetidasa Proteasomal , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Timo/citología , Timo/efectos de los fármacosRESUMEN
The AMP-activated protein kinase (AMPK) is related to a growing family of protein kinases that are believed to protect cells against environmental and nutritional stress. In the present study the hypothesis of a protective role for AMPK against thymocyte apoptosis has been tested. It is shown that AMPK is expressed in rat thymocytes that contain the transcript for the a1 isoform of the AMPK catalytic subunit and can be activated by treatment with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), a well-established activator of AMPK. AICAR is not toxic and prevents glucocorticoid-induced apoptosis in the same concentration range used to activate AMPK. At concentrations higher than 1 mM, AICAR fully restores cell viability and inhibits DNA laddering in dexamethasone-treated thymocytes. Furthermore, AICAR blocks the dexamethasone-induced activation of caspase 3-like enzymes, which are believed to play a pivotal role in apoptotic cell death. Activation of AMPK by oligomycin, which depletes thymocytes of ATP, is also correlated to inhibition of caspase 3-like activity in dexamethasone-treated cells. However, AICAR and oligomycin do not exert any protective action when apoptosis is induced by staurosporine. These results indicate that AICAR is a powerful inhibitor of glucocorticoid-induced apoptosis and suggest that AMPK activation may interfere with a step in the apoptotic cascade triggered by dexamethasone.
Asunto(s)
Aminoimidazol Carboxamida/análogos & derivados , Apoptosis/efectos de los fármacos , Caspasas , Glucocorticoides/farmacología , Complejos Multienzimáticos/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Ribonucleótidos/farmacología , Timo/citología , Proteínas Quinasas Activadas por AMP , Aminoimidazol Carboxamida/farmacología , Animales , Caspasa 3 , Supervivencia Celular , Cisteína Endopeptidasas/metabolismo , ADN/metabolismo , Dexametasona/farmacología , Activación Enzimática/efectos de los fármacos , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Complejos Multienzimáticos/genética , Reacción en Cadena de la Polimerasa , Proteínas Quinasas/genética , ARN/análisis , Ratas , Ratas Wistar , Timo/enzimologíaRESUMEN
Ceramide has emerged as a novel lipid mediator in cell growth and apoptosis. In difluoromethylornithine-resistant L1210 cells stimulated to growth from quiescence, the cell-permeant analogues of ceramide N-acetylsphingosine (C2-ceramide) and N-hexanoylsphingosine (C6-ceramide) inhibited the induction of ornithine decarboxylase (ODC) activity with IC50 of 8.3 and 1.5 microM respectively. This effect was strictly related to the ability to inhibit cell growth and [3H]thymidine incorporation. The suppression of cell growth was also associated with apoptosis. The addition of bacterial sphingomyelinase resulted in a significant, but limited, reduction of ODC induction and [3H]thymidine incorporation. Bacterial lipopolysaccharide, which may act as a ceramide analogue, also inhibited the induction of the enzyme. Moreover, C6-ceramide largely prevented the accumulation of ODC mRNA and its precursor, ODC heterogeneous nuclear RNA, that accompanied the induction of ODC activity. A slight increase in ODC turnover was also observed. The DNA-binding activity of some transcription factors known to bind and transactivate the ODC gene was investigated by gel mobility-shift assay under the same experimental conditions. However, only the binding of Myc/Max was negatively affected by the treatment with C6-ceramide. Furthermore, the amount of immunoreactive c-Myc, which increased after stimulation of the cells to growth, was strongly reduced by C6-ceramide. These results suggest that the inhibition of c-Myc and ODC expression may be early events in the response of leukaemia cells to ceramide.
Asunto(s)
Ceramidas/farmacología , Eflornitina/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes myc , Leucemia L1210/genética , Ornitina Descarboxilasa/genética , Animales , Permeabilidad de la Membrana Celular , Leucemia L1210/enzimología , Leucemia L1210/patología , Ratones , Células Tumorales CultivadasRESUMEN
In quiescent thymocytes, mitochondrial de-energization was not correlated to apoptotic death. In fact, thymocytes treated with oligomycin, a highly specific inhibitor of ATP synthase, alone or with atractyloside to block ATP translocation from the cytoplasm, were alive, even if their mitochondria were depolarized, as revealed by flow cytometry after Rhodamine 123 staining. Furthermore, oligomycin was a powerful inhibitor of apoptosis induced in rat thymocytes by dexamethasone and, to a lesser extent, by the calcium ionophore A23187 and etoposide, but was without effect when apoptosis was induced by staurosporine, and increased cell death in mitogen-treated thymocytes. The inhibition of apoptosis was confirmed by morphological criteria, inhibition of inter-nucleosomal DNA fragmentation and inhibition of the loss of membrane integrity. The anti-apoptotic effect of oligomycin in cells treated with A23187 or etoposide was correlated to the inhibition of protein synthesis, while inhibition of apoptosis induced by dexamethasone, already evident at an oligomycin concentration of 10 ng/ml, was instead strictly correlated to the effect exerted on the cellular ATP level. Thymocyte apoptosis triggered by dexamethasone was blocked or delayed by inhibitors of respiratory-chain uncouplers, inhibitors of ATP synthase and antioxidants: a lasting protection from dexamethasone-induced apoptosis was always correlated to a drastic and rapid reduction in ATP level (31-35% of control), while a delay in the death process was characterized by a moderate decrease in ATP (73-82% of control). Oligomycin inhibited the specific binding of radioactive corticosteroid to thymocyte nuclei, confirming the inhibitory effect of ATP depletion on glucocorticoid binding and suggesting that ATP depletion is a common mediator of the anti-apoptotic action of different effectors in glucocorticoid-induced apoptosis. In conclusion, the reported data indicate that ATP may act as a cellular modulator of some forms of apoptosis, depending on the death trigger, and that in quiescent cells the de-energization of mitochondria is not necessarily linked to apoptosis.
Asunto(s)
Adenosina Trifosfato/metabolismo , Antiinflamatorios/farmacología , Apoptosis/efectos de los fármacos , Dexametasona/farmacología , Linfocitos T/patología , Administración Tópica , Animales , Citometría de Flujo , Glucocorticoides , Masculino , Ratas , Ratas Wistar , Linfocitos T/metabolismoRESUMEN
In difluoromethylornithine-resistant L1210 cells stimulated to grow from quiescence, haloperidol caused an early and dose-dependent inhibition of the induction of ornithine decarboxylase (ODC) activity, with an IC50 of 3.5 microM. This effect was accompanied by a reduction in the ODC mRNA level and inhibition of cell growth. Other sigma ligands of different chemical classes inhibited the induction of ODC activity, whereas sulpiride, a dopamine antagonist devoid of sigma-binding affinity, was ineffective. These results indicate that the inhibition of ODC expression may be an early event involved in the antiproliferative response of leukemia cells to haloperidol.
Asunto(s)
Eflornitina/farmacología , Haloperidol/farmacología , Ornitina Descarboxilasa/biosíntesis , Ornitina Descarboxilasa/genética , Animales , Antineoplásicos/farmacología , Antipsicóticos/farmacología , División Celular/efectos de los fármacos , Resistencia a Medicamentos , Inducción Enzimática/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Leucemia L1210/enzimología , Leucemia L1210/genética , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
In difluoromethylornithine resistant L1210 cells stimulated to growth from quiescence, the selective kappa-opioidergic agonist trans-(+/-)-3,4-dichloro-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneaceta mid e (U-50488H) caused a dose dependent inhibition of the induction of ODC activity, with a half-maximal effect at about 1 microM. U-50488H also provoked reduction of ODC mRNA level and increase of ODC turnover, as well as inhibition of cell growth. U-69593, another kappa-selective agonist, was only slightly effective. The action of U-50488H on ODC induction was not blocked by naloxone, beta-chlornaltrexamine or by the kappa-selective opioid antagonists Mr1452 and nor-binaltorphimine (nBNI). Actually Mr1452 and nBNI exerted some inhibitory effect. Furthermore, the separated enantiomers (+) and (-) of U-50488H were similarly effective. The (-)cis-(1S,2R)-U50488 stereoisomer, exhibiting low affinity for kappa and high affinity for sigma receptors and carbetapentane, another sigma ligand, also inhibited ODC induction, although less effectively than U-50488H. None of several other opioid ligands tested had significant effects on ODC induction. In conclusion, the inhibition of ODC expression by U-50488H does not involve classical, enantiospecific opioid receptors; rather, these results suggest the involvement of a distinct site of action linked to inhibition of lymphoid cell proliferation.
Asunto(s)
Bencenoacetamidas , Eflornitina/farmacología , Linfocitos/enzimología , Antagonistas de Narcóticos/farmacología , Ornitina Descarboxilasa/biosíntesis , Pirrolidinas/farmacología , Receptores Opioides kappa/metabolismo , 3,4-Dicloro-N-metil-N-(2-(1-pirrolidinil)-ciclohexil)-bencenacetamida, (trans)-Isómero , Animales , Secuencia de Bases , División Celular/efectos de los fármacos , Cartilla de ADN/química , Resistencia a Medicamentos , Inducción Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Leucemia L1210 , Linfocitos/citología , Linfocitos/efectos de los fármacos , Ratones , Datos de Secuencia Molecular , Narcóticos/metabolismo , Narcóticos/farmacología , Ornitina Descarboxilasa/genética , ARN Mensajero/análisis , Receptores Opioides kappa/agonistas , Estereoisomerismo , Células Tumorales CultivadasRESUMEN
Internucleosomal DNA fragmentation and cell death induced by dexamethasone in rat thymocytes were inhibited when cells were cultured in 95% N2/5% CO2 atmosphere, in which oxygen was rapidly reduced to under 0.5%. DNA fragmentation was delayed by a less severe hypoxia in 5% oxygen whilst in cell cultured in high oxygen atmosphere (95% O2) cell death was increased. On the other hand, prolonged oxygen deprivation caused an increase of spontaneous apoptotic cell death. Hypoxia also inhibited DNA fragmentation induced by calcium ionophore A23187, but not by topoisomerase inhibitor camptothecin. These data support the hypothesis of the involvement of oxygen reactive species in calcium-mediated apoptosis and suggest a complex role of oxygen in the modulation of programmed cell death.
Asunto(s)
Apoptosis/efectos de los fármacos , ADN/metabolismo , Dexametasona/farmacología , Oxígeno/farmacología , Timo/citología , Animales , Calcimicina/farmacología , ADN/efectos de los fármacos , Electroforesis en Gel de Agar , L-Lactato Deshidrogenasa/metabolismo , Masculino , Microscopía Electrónica , Oxígeno/administración & dosificación , Ratas , Ratas Wistar , Timo/efectos de los fármacos , Factor de Transcripción AP-1/metabolismo , Proteína p53 Supresora de Tumor/fisiologíaRESUMEN
Coenzyme Q10 (CoQ10) plays an essential physiologic role in oxidative phosphorylation and its plasma and tissue concentration has been evaluated in various pathologic conditions, both endocrine and non endocrine; among the latter particularly in cardiac failure. Plasma CoQ10 determination has been reported in the literature an a useful diagnostic tool in differential diagnosis of thyroid diseases. In the present study we have evaluated CoQ10 circulating levels both in hypo- and hyperthyroidism. For this purpose plasma CoQ10, fT3-fT4 and TSH concentrations have been determined (HPLC, RIA and IRMA respectively) in a group of hypothyroid patients, hyperthyroid and control subjects. No patient was harbouring cardiovascular, metabolic or systemic disease. CoQ10 has resulted 0.97 +/- 0.46 mcg/ml in the hypothyroid group, 0.51 +/- 0.35 in hyperthyroid and 0.73 +/- 0.16 in control group, with a significative difference between first and second group only; more, the prevalence of high levels has appeared greater in hypo- towards hyperthyroid patients and that of low levels in the latter greater than in the former. Finally an inverse relation of CoQ10 with fT3 and tT3, but not with fT4 and tT4, has been shown. In conclusion, plasma CoQ10 levels have not given in this study a sharp distinction between euthyroidism on a side and hypo- and hyperthyroidism on the other, but necessity of longitudinal studies after therapy is outlined, both to know time of normalization of plasma concentrations and to verify the opportunity of exogenous administration of CoQ10 in hyperthyroid patients with risk factors for heart failure.